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Previous metabolic studies have demonstrated that leishmania parasites are able to synthesise proline from glutamic acid and threonine from aspartic acid. The first committed step in both biosynthetic pathways involves an amino acid kinase, either a glutamate 5‐kinase (G5K; http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/2/11.html) or an aspartokinase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/2/4.html). Bioinformatic analysis of multiple leishmania genomes identifies a single amino acid‐kinase gene (LdBPK 262740.1) variously annotated as either a putative glutamate or aspartate kinase. To establish the catalytic function of this Leishmania donovani gene product, we have determined the physical and kinetic properties of the recombinant enzyme purified from Escherichia coli. The findings indicate that the enzyme is a bona fide G5K with no activity as an aspartokinase. Tetrameric G5K displays kinetic behaviour similar to its bacterial orthologues and is allosterically regulated by proline, the end product of the pathway. The structure‐activity relationships of proline analogues as inhibitors are broadly similar to the bacterial enzyme. However, unlike G5K from E. coli, leishmania G5K lacks a C‐terminal PUA (pseudouridine synthase and archaeosine transglycosylase) domain and does not undergo higher oligomerisation in the presence of proline. Gene replacement studies are suggestive, but not conclusive that G5K is essential. Enzymes Glutamate 5‐kinase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/2/11.html); aspartokinase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/2/4.html).

Significance Malaria and cryptosporidiosis are major burdens to both global health and economic development in many countries. Malaria caused >400,000 deaths in 2017, and cryptosporidiosis is estimated to cause >200,000 deaths a year. The spread of drug resistance is a growing concern for malaria treatment, and there is no effective treatment for malnourished or immunocompromised children infected with Cryptosporidium. New treatments with novel mechanisms of action are needed for both diseases. We present a selective inhibitor of both Plasmodium and Cryptosporidium lysyl-tRNA synthetase capable of clearing parasites from mouse models of malaria and cryptosporidiosis infection. This provides very strong validation of lysyl-tRNA synthetase as a drug target in these organisms and a lead for further drug discovery., Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Plasmodium falciparum (Pf) prolyl-tRNA synthetase (ProRS) is one of the few chemical-genetically validated drug targets for malaria, yet highly selective inhibitors have not been described. In this paper, approximately 40,000 compounds were screened to identify compounds that selectively inhibit PfProRS enzyme activity versus Homo sapiens (Hs) ProRS. X-ray crystallography structures were solved for apo, as well as substrate- and inhibitor-bound forms of PfProRS. We identified two new inhibitors of PfProRS that bind outside the active site. These two allosteric inhibitors showed >100 times specificity for PfProRS compared to HsProRS, demonstrating this class of compounds could overcome the toxicity related to HsProRS inhibition by halofuginone and its analogues. Initial medicinal chemistry was performed on one of the two compounds, guided by the cocrystallography of the compound with PfProRS, and the results can instruct future medicinal chemistry work to optimize these promising new leads for drug development against malaria.

International audience; Les kinétoplastidés sont des protozoaires flagellés responsables de maladies tropicales négligées mortelles telles que la leishmaniose viscérale (L. donovani et L. infantum) ou la trypanosomiase africaine (T. brucei), auxquelles plus de 500 millions de personnes sont exposées et pour lesquelles les traitements disponibles sont très limités [1,2]. Depuis quelques années, on observe un regain d’intérêt pour le développement de nitrohétérocycles anti-infectieux tels que le delamanide et le fexinidazole [3,4]. De récentes études indiquent que l’activité anti-kinétoplastidés de ces dérivés repose sur leur réduction sélective par des nitroréductases parasitaires (NTR1 et NTR2 chez Leishmania, NTR chez Trypanosoma), conduisant à la formation de métabolites cytotoxiques [5-7].Suite à des travaux préliminaires réalisés dans notre équipe en série 8-nitroquinoléin-2(1H)-ones [8-10], nous présentons ici la synthèse de 60 nouveaux hétérocycles nitro-aromatiques, l’étude de leurs propriétés physico-chimiques (incluant les potentiels de réduction) et pharmacocinétiques in vitro (stabilité microsomale et fixation aux protéines plasmatiques), de leur activité anti-kinétoplastidés in vitro de même que la recherche de leur mécanisme d’action et l’étude de leur cytotoxicité et génotoxicité. Ainsi, ce travail nous a permis d’identifier 3 nouveaux hits (2 anti-kinétoplastidés et 1 sélectif de Trypanosoma), d’observer une corrélation entre les potentiels de réduction et l’activité antileishmanienne, de déterminer que ces molécules sont sélectivement bioactivées par la NTR1 chez L. donovani et qu’elles ne sont pas génotoxiques vis-à-vis de cellules humaines. Dans ce contexte, des études in vivo sur un modèle murin de trypanosomiase sont en cours dans le but de statuer sur le devenir de ces molécules en tant que candidats antiparasitaires humains ou vétérinaires.Références

European Commission FP7 Marie Curie Initial Training Network ‘ParaMet’ [290080 to S.S.]; ANR project MetaboHub [ANR-11-INBS-0010 to B.M.]; Wellcome Trust [085349]; The work of Fiona Achcar was part of the SysMO SilicoTryp project coordinated by R.B. Funding for open access charge: European Commission FP7 Marie Curie Initial Training Network ‘ParaMet’ [290080]. The metabolic network of a cell represents the catabolic and anabolic reactions that interconvert small molecules (metabolites) through the activity of enzymes, transporters and non-catalyzed chemical reactions. Our understanding of individual metabolic networks is increasing as we learn more about the enzymes that are active in particular cells under particular conditions and as technologies advance to allow detailed measurements of the cellular metabolome. Metabolic network databases are of increasing importance in allowing us to contextualise data sets emerging from transcriptomic, proteomic and metabolomic experiments. Here we present a dynamic database, TrypanoCyc (http://www.metexplore.fr/trypanocyc/), which describes the generic and condition-specific metabolic network of Trypanosoma brucei, a parasitic protozoan responsible for human and animal African trypanosomiasis. In addition to enabling navigation through the BioCyc-based TrypanoCyc interface, we have also implemented a network-based representation of the information through MetExplore, yielding a novel environment in which to visualise the metabolism of this important parasite. Publisher PDF

De novo synthesis of threonine from aspartate occurs via the β-aspartyl phosphate pathway in plants, bacteria and fungi. However, the Trypanosoma brucei genome encodes only the last two steps in this pathway: homoserine kinase (HSK) and threonine synthase. Here, we investigated the possible roles for this incomplete pathway through biochemical, genetic and nutritional studies. Purified recombinant TbHSK specifically phosphorylates L-homoserine and displays kinetic properties similar to other HSKs. HSK null mutants generated in bloodstream forms displayed no growth phenotype in vitro or loss of virulence in vivo. However, following transformation into procyclic forms, homoserine, homoserine lactone and certain acyl homoserine lactones (AHLs) were found to substitute for threonine in growth media for wild-type procyclics, but not HSK null mutants. The tsetse fly is considered to be an unlikely source of these nutrients as it feeds exclusively on mammalian blood. Bioinformatic studies predict that tsetse endosymbionts possess part (up to homoserine in Wigglesworthia glossinidia) or all of the β-aspartyl phosphate pathway (Sodalis glossinidius). In addition S. glossinidius is known to produce 3-oxohexanoylhomoserine lactone which also supports trypanosome growth. We propose that T. brucei has retained HSK and threonine synthase in order to salvage these nutrients when threonine availability is limiting.

Trypanosoma brucei N-myristoyltransferase (TbNMT) is an attractive therapeutic target for the treatment of human African trypanosomiasis (HAT). From previous studies, we identified pyrazole sulfonamide, DDD85646 (1), a potent inhibitor of TbNMT. Although this compound represents an excellent lead, poor central nervous system (CNS) exposure restricts its use to the hemolymphatic form (stage 1) of the disease. With a clear clinical need for new drug treatments for HAT that address both the hemolymphatic and CNS stages of the disease, a chemistry campaign was initiated to address the shortfalls of this series. This paper describes modifications to the pyrazole sulfonamides which markedly improved blood-brain barrier permeability, achieved by reducing polar surface area and capping the sulfonamide. Moreover, replacing the core aromatic with a flexible linker significantly improved selectivity. This led to the discovery of DDD100097 (40) which demonstrated partial efficacy in a stage 2 (CNS) mouse model of HAT.

Co- and post-translational N-myristoylation is known to play a role in the correct subcellular localization of specific proteins in eukaryotes. The enzyme that catalyses this reaction, NMT (N-myristoyltransferase), has been pharmacologically validated as a drug target in the African trypanosome, Trypanosoma brucei. In the present study, we evaluate NMT as a potential drug target in Trypanosoma cruzi, the causative agent of Chagas’ disease, using chemical and genetic approaches. Replacement of both allelic copies of TcNMT (T. cruzi NMT) was only possible in the presence of a constitutively expressed ectopic copy of the gene, indicating that this gene is essential for survival of T. cruzi epimastigotes. The pyrazole sulphonamide NMT inhibitor DDD85646 is 13–23-fold less potent against recombinant TcNMT than TbNMT (T. brucei NMT), with Ki values of 12.7 and 22.8 nM respectively, by scintillation proximity or coupled assay methods. DDD85646 also inhibits growth of T. cruzi epimastigotes (EC50=6.9 μM), but is ~1000-fold less potent than that reported for T. brucei. On-target activity is demonstrated by shifts in cell potency in lines that over- and under-express NMT and by inhibition of intracellular N-myristoylation of several proteins in a dose-dependent manner. Collectively, our findings suggest that N-myristoylation is an essential and druggable target in T. cruzi., The present study shows that N-myristoyltransferase is essential for growth of Trypanosoma cruzi, the parasite responsible for Chagas’ disease. The kinetic properties of the enzyme are described along with evidence that growth is specifically inhibited by blocking N-myristoylation in the parasite.

Formation of Bacillus subtilis biofilms, consisting of cells encapsulated within an extracellular matrix of exopolysaccharide and protein, requires the polyamine spermidine. A recent study reported that (1) related polyamine norspermidine is synthesized by B. subtilis using the equivalent of the Vibrio cholerae biosynthetic pathway, (2) exogenous norspermidine at 25 μM prevents B. subtilis biofilm formation, (3) endogenous norspermidine is present in biofilms at 50-80 μM, and (4) norspermidine prevents biofilm formation by condensing biofilm exopolysaccharide. In contrast, we find that, at concentrations up to 200 μM, exogenous norspermidine promotes biofilm formation. We find that norspermidine is absent in wild-type B. subtilis biofilms at all stages, and higher concentrations of exogenous norspermidine eventually inhibit planktonic growth and biofilm formation in an exopolysaccharide-independent manner. Moreover, orthologs of the V. cholerae norspermidine biosynthetic pathway are absent from B. subtilis, confirming that norspermidine is not physiologically relevant to biofilm function in this species.

Bifunctional dihydrofolate reductase–thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed., Author Summary There are few validated and fully characterised targets suitable for drug discovery against African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the biochemical properties of the bifunctional enzyme, dihydrofolate reductase–thymidylate synthase (DHFR-TS), and its susceptibility to a range of classical inhibitors normally used in the treatment of cancer, bacterial or protozoal infections. Some of these drugs are extremely potent against the isolated enzyme, but much less so against the intact trypanosome. We have found that modulating certain medium components can affect drug sensitivity, presumably by either competition for uptake and competition for the active site of DHFR-TS. In the case of one human TS inhibitor (raltitrexed) the inhibitor is more potent against the intact parasite. We propose that addition of extra glutamic acid residues not only improves retention in the cell, but also increases potency against TS, as it does in human cells.

In this paper we describe the optimization of a phenotypic hit against Plasmodium falciparum, based on a trisubstituted pyrimidine scaffold. This led to compounds with good pharmacokinetics and oral activity in a P. berghei mouse model of malaria. The most promising compound (13) showed a reduction in parasitemia of 96% when dosed at 30 mg/kg orally once a day for 4 days in the P. berghei mouse model of malaria. It also demonstrated a rapid rate of clearance of the erythrocytic stage of P. falciparum in the SCID mouse model with an ED90 of 11.7 mg/kg when dosed orally. Unfortunately, the compound is a potent inhibitor of cytochrome P450 enzymes, probably due to a 4-pyridyl substituent. Nevertheless, this is a lead molecule with a potentially useful antimalarial profile, which could either be further optimized or be used for target hunting.

SCYX-7158, an oxaborole, is currently in Phase I clinical trials for the treatment of human African trypanosomiasis. Here we investigate possible modes of action against Trypanosoma brucei using orthogonal chemo-proteomic and genomic approaches. SILAC-based proteomic studies using an oxaborole analogue immobilised onto a resin was used either in competition with a soluble oxaborole or an immobilised inactive control to identify thirteen proteins common to both strategies. Cell-cycle analysis of cells incubated with sub-lethal concentrations of an oxaborole identified a subtle but significant accumulation of G2 and >G2 cells. Given the possibility of compromised DNA fidelity, we investigated long-term exposure of T. brucei to oxaboroles by generating resistant cell lines in vitro. Resistance proved more difficult to generate than for drugs currently used in the field, and in one of our three cell lines was unstable. Whole-genome sequencing of the resistant cell lines revealed single nucleotide polymorphisms in 66 genes and several large-scale genomic aberrations. The absence of a simple consistent mechanism among resistant cell lines and the diverse list of binding partners from the proteomic studies suggest a degree of polypharmacology that should reduce the risk of resistance to this compound class emerging in the field. The combined genetic and chemical biology approaches have provided lists of candidates to be investigated for more detailed information on the mode of action of this promising new drug class., Author Summary The mode of action of a new class of boron-containing chemicals (the oxaboroles), currently under development for the treatment of human African trypanosomiasis, is unknown. Here we identify a number of potential candidate proteins that could be involved either in the mode of action of these compounds or in the mechanism of resistance. This information could prove critical in protecting the compounds against resistance emerging in the field as well as opening up new avenues for drug discovery.

Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines ((oe)RNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the (oe)RNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis.

Glutathionylspermidine is an intermediate formed in the biosynthesis of trypanothione, an essential metabolite in defence against chemical and oxidative stress in the Kinetoplastida. The kinetic mechanism for glutathionylspermidine synthetase (EC 6.3.1.8) from Crithidia fasciculata (CfGspS) obeys a rapid equilibrium random ter-ter model with kinetic constants K(GSH) = 609 microM, K(Spd) = 157 microM and K(ATP) = 215 microM. Phosphonate and phosphinate analogues of glutathionylspermidine, previously shown to be potent inhibitors of GspS from Escherichia coli, are equally potent against CfGspS. The tetrahedral phosphonate acts as a simple ground state analogue of glutathione (GSH) (K(i) approximately 156 microM), whereas the phosphinate behaves as a stable mimic of the postulated unstable tetrahedral intermediate. Kinetic studies showed that the phosphinate behaves as a slow-binding bisubstrate inhibitor [competitive with respect to GSH and spermidine (Spd)] with rate constants k(3) (on rate) = 6.98 x 10(4) M(-1) x s(-1) and k(4) (off rate) = 1.3 x 10(-3) s(-1), providing a dissociation constant K(i) = 18.6 nM. The phosphinate analogue also inhibited recombinant trypanothione synthetase (EC 6.3.1.9) from C. fasciculata, Leishmania major, Trypanosoma cruzi and Trypanosoma brucei with K(i)(app) values 20-40-fold greater than that of CfGspS. This phosphinate analogue remains the most potent enzyme inhibitor identified to date, and represents a good starting point for drug discovery for trypanosomiasis and leishmaniasis.

There is an urgent need for new drugs to treat malaria, with broad therapeutic potential and novel modes of action, to widen the scope of treatment and to overcome emerging drug resistance. Here we describe the discovery of DDD107498, a compound with a potent and novel spectrum of antimalarial activity against multiple life-cycle stages of the Plasmodium parasite, with good pharmacokinetic properties and an acceptable safety profile. DDD107498 demonstrates potential to address a variety of clinical needs, including single-dose treatment, transmission blocking and chemoprotection. DDD107498 was developed from a screening programme against blood-stage malaria parasites; its molecular target has been identified as translation elongation factor 2 (eEF2), which is responsible for the GTP-dependent translocation of the ribosome along messenger RNA, and is essential for protein synthesis. This discovery of eEF2 as a viable antimalarial drug target opens up new possibilities for drug discovery.

Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Because this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidized form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 A, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through pi-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with nonplanar molecules, such as clomipramine, where stacking is not possible.